Polypeptide-vaccines for broad protection against hypervirulent meningococcal lineages

ABSTRACT

A small number of defined antigens can provide broad protection against meningococcal infection, and the invention provides a composition which, after administration to a subject, is able to induce an antibody response in that subject, wherein the antibody response is bactericidal against two or three of hypervirulent lineages A4, ET 5 and lineage 3 of  N. meningitidis  serogroup B. Rather than consisting of a single antigen, the composition comprises a mixture of 10 or fewer purified antigens, and should not include complex or undefined mixtures of antigens such as outer membrane vesicles. Five protein antigens are used in particular: (1) a ‘NadA’ protein; (2) a ‘741’ protein; (3) a ‘936’ protein; (4) a ‘953’ protein; and (5) a ‘287’ protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of Ser. No. 10/530,753, filed Mar. 3,2006, which is the National Phase of PCT Application PCT/IB03/04848,filed Oct. 2, 2003, which claims the benefit of GB Application0309115.4, filed Apr. 22, 2003, GB Application 0305831.0, filed Mar. 13,2003, and GB Application 0223741.0, filed Oct. 11, 2002, all of whichare incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention is in the fields of immunology and vaccinology. Inparticular, it relates to antigens from Neisseria meningitidis(meningococcus) and their use in immunization.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 223002100301SeqList.txt,date recorded: Jan. 30, 2014, size: 31 KB).

BACKGROUND

N. meningitidis is a non-motile, Gram-negative human pathogen thatcolonizes the pharynx and causes meningitis (and, occasionally,septicemia in the absence of meningitis). It causes both endemic andepidemic disease. Following the introduction of the conjugate vaccineagainst Haemophilus influenzae, N. meningitidis is the major cause ofbacterial meningitis in the USA.

Based on the organism's capsular polysaccharide, various serogroups ofN. meningitidis have been identified. Serogroup A is the pathogen mostoften implicated in epidemic disease in sub-Saharan Africa. Serogroups Band C are responsible for the vast majority of cases in the UnitedStates and in most developed countries. Serogroups W135 and Y areresponsible for the rest of the cases in the USA and developedcountries. After serogroup, classification includes serotype,serosubtype and then immunotype, and the standard nomenclature listsserogroup, serotype, serosubtype, and immunotype, each separated by acolon e.g. B:4:P1.15:L3,7,9. Within serogroup B, some lineages causedisease often (hyperinvasive), some lineages cause more severe forms ofdisease than others (hypervirulent), and others rarely cause disease atall. Seven hypervirulent lineages are recognized, namely subgroups I,III and IV-1, ET-5 complex, ET-37 complex, A4 cluster and lineage 3.These have been defined by multilocus enzyme electrophoresis (MLEE), butmultilocus sequence typing (MLST) has also been used to classifymeningococci [ref 1].

A polysaccharide vaccine against serogroups A, C, W135 & Y has beenknown for many years [2, 3] but a vaccine against serogroup B has provedelusive. Vaccines based on outer-membrane vesicles have been tested[e.g. see ref. 4], but the protection afforded by these vaccines istypically restricted to the strain used to make the vaccine. Thereremains a need, therefore, for a broadly-effective serogroup B vaccine.

Genome sequences for meningococcal serogroups A [5] and B [6,7] havebeen reported, and the serogroup B sequence has been studied to identifyvaccine antigens [e.g. refs. 8 to 13]. Candidate antigens have beenmanipulated to improve heterologous expression [refs. 14 to 16].

It is an object of the invention to provide further and improvedcompositions for providing immunity against meningococcal disease and/orinfection, and in particular for providing broad immunity againstserogroup B meningococcus.

DISCLOSURE OF THE INVENTION

Vaccines against pathogens such as hepatitis B virus, diphtheria andtetanus typically contain a single protein antigen (e.g. the HBV surfaceantigen, or a tetanus toxoid). In contrast, acellular whooping coughvaccines typically contain at least three B. pertussis proteins and thePrevenar™ pneumococcal vaccine contains seven separate conjugatedsaccharide antigens. Other vaccines such as cellular pertussis vaccines,the measles vaccine, the inactivated polio vaccine (IPV) andmeningococcal OMV vaccines are by their very nature complex mixtures ofa large number of antigens.

Whether protection against can be elicited by a single antigen, a smallnumber of defined antigens, or a complex mixture of undefined antigens,therefore depends on the pathogen in question. The invention is based onthe discovery that a small number of defined antigens is able to providebroad protection against meningococcal infection, and the inventionprovides a composition which, after administration to a subject, is ableto induce an antibody response in that subject, wherein the antibodyresponse is bactericidal against two or more (e.g. 2 or 3) ofhypervirulent lineages A4, ET-5 and lineage 3 of N. meningitidisserogroup B.

Rather than consisting of a single antigen, it is preferred that thecomposition of the invention comprises a mixture of 10 or fewer (e.g. 9,8, 7, 6, 5, 4, 3, 2) purified antigens, and it is particularly preferredthat the composition should not include complex or undefined mixtures ofantigens e.g. it is preferred not to include outer membrane vesicles inthe composition.

For serogroup B meningococcus, a mixture of five defined proteinantigens has been found to elicit a good protective immune response. Theinvention thus provides a composition comprising the following fivemeningococcal protein antigens: (1) a ‘NadA’ protein; (2) a ‘741’protein; (3) a ‘936’ protein; (4) a ‘953’ protein; and (5) a ‘287’protein. These antigens are referred to herein as the ‘five basicantigens’.

NadA Protein

‘NadA’ (Neisserial adhesin A) from serogroup B of N. meningitidis isdisclosed as protein ‘961’ in reference 10 (SEQ IDs 2943 & 2944) and as‘NMB1994’ in reference 6 (see also GenBank accession numbers: 11352904 &7227256). A detailed description of the protein can be found inreference 17. There is no corresponding protein in serogroup A [5, 17].

When used according to the present invention, NadA may take variousforms. Preferred forms of NadA are truncation or deletion variants, suchas those disclosed in references 14 to 16. In particular, NadA withoutits C-terminal membrane anchor is preferred (e.g. deletion of residues351-405 for strain 2996 [SEQ ID 1]), which is sometimes distinguishedherein by the use of a ‘C’ superscript e.g. NadA^((c)). Expression ofNadA without its membrane anchor domain (e.g. SEQ ID 1) in E. coliresults in secretion of the protein into the culture supernatant withconcomitant removal of its 23 mer leader peptide (e.g. to leave a 327mer for strain 2996 [SEQ ID 2]). Polypeptides without their leaderpeptides are sometimes distinguished herein by the use of a ‘NL’superscript e.g. NadA^((NL)) or NadA^((C)(NL)).

Preferred NadA sequences have 50% or more identity (e.g. 60%, 70%, 80%,90%, 95%, 99% or more) to SEQ ID 2. This includes NadA variants (e.g.allelic variants, homologs, orthologs, paralogs, mutants, etc.). Allelicforms of NadA are shown in FIG. 9 of reference 18.

Other preferred NadA sequences comprise at least n consecutive aminoacids from SEQ ID 1, wherein n is 7 or more (eg. 8, 10, 12, 14, 16, 18,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).Preferred fragments comprise an epitope from NadA. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or the N-terminus of SEQID 1 (e.g. NadA^((c)), NadA^((NL)), NadA^((c)(NL)). Where N-terminusresidues are deleted, it is preferred that the deletion should notremove the ability of NadA to adhere to human epithelial cells. Apreferred fragment of SEQ ID 1 is SEQ ID 2.

Secreted NadA can conveniently be prepared in highly pure form fromculture supernatant by a process comprising the steps of: concentrationand diafiltration against a buffer by ultrafiltration; anionic columnchromatography; hydrophobic column chromatography; hydroxylapatiteceramic column chromatography; diafiltration against a buffer; andfilter sterilisation. Further details of the process are given in theexamples.

NadA is preferably used in an oligomeric form (e.g. in trimeric form).

741 Protein

‘741’ protein from serogroup B is disclosed in reference 10 (SEQ IDs2535 & 2536) and as ‘NMB 1870’ in reference 6 (see also GenBankaccession number GI:7227128). The corresponding protein in serogroup A[5] has GenBank accession number 7379322. 741 is naturally alipoprotein.

When used according to the present invention, 741 protein may takevarious forms. Preferred forms of 741 are truncation or deletionvariants, such as those disclosed in references 14 to 16. In particular,the N-terminus of 741 may be deleted up to and including itspoly-glycine sequence (i.e. deletion of residues 1 to 72 for strain MC58[SEQ ID 3]), which is sometimes distinguished herein by the use of a‘ΔG’ prefix. This deletion can enhance expression. The deletion alsoremoves 741's lipidation site.

Preferred 741 sequences have 50% or more identity (e.g. 60%, 70%, 80%,90%, 95%, 99% or more) to SEQ ID 3. This includes 741 variants (e.g.allelic variants, homologs, orthologs, paralogs, mutants, etc.). Allelicforms of 741 can be found in SEQ IDs 1 to 22 of reference 16, and in SEQIDs 1 to 23 of reference 19. SEQ IDs 1-299 of reference 20 give further741 sequences.

Other preferred 741 sequences comprise at least n consecutive aminoacids from SEQ ID 3, wherein n is 7 or more (eg. 8, 10, 12, 14, 16, 18,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).Preferred fragments comprise an epitope from 741. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or the N-terminus of SEQID 3.

Protein 741 is an extremely effective antigen for elicitinganti-meningococcal antibody responses, and it is expressed across allmeningococcal serogroups. Phylogenetic analysis shows that the proteinsplits into two groups, and that one of these splits again to give threevariants in total [21], and while serum raised against a given variantis bactericidal within the same variant group, it is not active againststrains which express one of the other two variants i.e. there isintra-variant cross-protection, but not inter-variant cross-protection.For maximum cross-strain efficacy, therefore, it is preferred that acomposition should include more than one variant of protein 741. Anexemplary sequence from each variant is given in SEQ ID 10, 11 and 12herein, starting with a N-terminal cysteine residue to which a lipidwill be covalently attached in the lipoprotein form of 741.

It is therefore preferred that the composition should include at leasttwo of (1) a first protein, comprising an amino acid sequence having atleast a % sequence identity to SEQ ID 10 and/or comprising an amino acidsequence consisting of a fragment of at least x contiguous amino acidsfrom SEQ ID 10; (2) a second protein, comprising an amino acid sequencehaving at least b % sequence identity to SEQ ID 11 and/or comprising anamino acid sequence consisting of a fragment of at least y contiguousamino acids from SEQ ID 11; and (3) a third protein, comprising an aminoacid sequence having at least c % sequence identity to SEQ ID 12 and/orcomprising an amino acid sequence consisting of a fragment of at least zcontiguous amino acids from SEQ ID 12.

The value of a is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.5, or more. The value of b is at least 85 e.g.86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more.The value of c is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.5, or more. The values of a, b and c are notintrinsically related to each other.

The value of x is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of y isat least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,120, 140, 160, 180, 200, 225, 250). The value of z is at least 7 e.g. 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 225, 250). The values of x, y and z are not intrinsically relatedto each other.

It is preferred that any given 741 amino acid sequence will not fallinto more than one of categories (1), (2) and (3). Any given 741sequence will thus fall into only one of categories (1), (2) and (3). Itis thus preferred that: protein (1) has less than 1% sequence identityto protein (2); protein (1) has less than j % sequence identity toprotein (3); and protein (2) has less than ex, sequence identity toprotein (3). The value of i is 60 or more (e.g. 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, etc.) and is at most a. The value of j is 60 ormore (e.g. 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, etc.) and isat most b. The value of k is 60 or more (e.g. 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, etc.) and is at most c. The values oft, j and kare not intrinsically related to each other.

936 Protein

‘936’ protein from serogroup B is disclosed in reference 10 (SEQ IDs2883 & 2884) and as ‘NMB2091’ in reference 6 (see also GenBank accessionnumber GI:7227353). The corresponding gene in serogroup A [5] hasGenBank accession number 7379093.

When used according to the present invention, 936 protein may takevarious forms. Preferred forms of 936 are truncation or deletionvariants, such as those disclosed in references 14 to 16. In particular,the N-terminus leader peptide of 936 may be deleted (i.e. deletion ofresidues 1 to 23 for strain MC58 [SEQ ID 4]) to give 936^((NL)).

Preferred 936 sequences have 50% or more identity (e.g. 60%, 70%, 80%,90%, 95%, 99% or more) to SEQ ID 4. This includes variants (e.g. allelicvariants, homologs, orthologs, paralogs, mutants etc).

Other preferred 936 sequences comprise at least n consecutive aminoacids from SEQ ID 4, wherein n is 7 or more (eg. 8, 10, 12, 14, 16, 18,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).Preferred fragments comprise an epitope from 936. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or the N-terminus of SEQID 4.

953 Protein

‘953’ protein from serogroup B is disclosed in reference 10 (SEQ IDs2917 & 2918) and as ‘NMB1030’ in reference 6 (see also GenBank accessionnumber GI:7226269). The corresponding protein in serogroup A [5] hasGenBank accession number 7380108.

When used according to the present invention, 953 protein may takevarious forms. Preferred forms of 953 are truncation or deletionvariants, such as those disclosed in references 14 to 16. In particular,the N-terminus leader peptide of 953 may be deleted (i.e. deletion ofresidues 1 to 19 for strain MC58 [SEQ ID 5]) to give 953^((NL)).

Preferred 953 sequences have 50% or more identity (e.g. 60%, 70%, 80%,90%, 95%, 99% or more) to SEQ ID 5. This includes 953 variants (e.g.allelic variants, homologs, orthologs, paralogs, mutants, etc.). Allelicforms of 953 can be seen in FIG. 19 of reference 12.

Other preferred 953 sequences comprise at least n consecutive aminoacids from SEQ ID 5, wherein n is 7 or more (eg. 8, 10, 12, 14, 16, 18,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).Preferred fragments comprise an epitope from 953. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or the N-terminus of SEQID 5.

287 Protein

‘287’ protein from serogroup B is disclosed in reference 10 (SEQ JDs3103 & 3104), as ‘NMB2132’ in reference 6, and as ‘GNA2132’ in reference13 (see also GenBank accession number GI:7227388). The correspondingprotein in serogroup A [5] has GenBank accession number 7379057.

When used according to the present invention, 287 protein may takevarious forms. Preferred forms of 287 are truncation or deletionvariants, such as those disclosed in references 14 to 16. In particular,the N-terminus of 287 may be deleted up to and including itspoly-glycine sequence (i.e. deletion of residues 1 to 24 for strain MC58[SEQ ID 6]), which is sometimes distinguished herein by the use of a‘ΔG’ prefix. This deletion can enhance expression.

Preferred 287 sequences have 50% or more identity (e.g. 60%, 70%, 80%,90%, 95%, 99% or more) to SEQ ID 6. This includes 287 variants (e.g.allelic variants, homologs, orthologs, paralogs, mutants, etc.). Allelicforms of 287 can be seen in FIGS. 5 and 15 of reference 12, and inexample 13 and FIG. 21 of reference 10 (SEQ IDs 3179 to 3184).

Other preferred 287 sequences comprise at least n consecutive aminoacids from SEQ ID 6, wherein n is 7 or more (eg. 8, 10, 12, 14, 16, 18,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more).Preferred fragments comprise an epitope from 287. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or the N-terminus of SEQID 6.

Fusion Proteins

The five antigens may be present in the composition as five separateproteins, but it is preferred that at least two of the antigens areexpressed as a single polypeptide chain (a ‘hybrid’ protein [refs. 14 to16]) e.g. such that the five antigens form fewer than five polypeptides.Hybrid proteins offer two principal advantages: first, a protein thatmay be unstable or poorly expressed on its own can be assisted by addinga suitable hybrid partner that overcomes the problem; second, commercialmanufacture is simplified as only one expression and purification needbe employed in order to produce two separately-useful proteins.

A hybrid protein included in a composition of the invention may comprisetwo or more (i.e. 2, 3, 4 or 5) of the five basic antigens. Hybridsconsisting of two of the five basic antigens are preferred.

Within the combination of five basic antigens, an antigen may be presentin more than one hybrid protein and/or as a non-hybrid protein. It ispreferred, however, that an antigen is present either as a hybrid or asa non-hybrid, but not as both, although it may be useful to includeprotein 741 both as a hybrid and a non-hybrid (preferably lipoprotein)antigen, particularly where more than one variant of 741 is used.

Two-antigen hybrids for use in the invention comprise: NadA & 741; NadA& 936; NadA & 953; NadA & 287; 741 & 936; 741 & 953; 741 & 287; 936 &953; 936 & 287; 953 & 287. Preferred two-antigen hybrids comprise: 741 &936; 953 & 287.

Hybrid proteins can be represented by the formulaNH₂-A-[-X-L-]_(n)-B—COOH, wherein: X is an amino acid sequence of one ofthe five basic antigens; L is an optional linker amino acid sequence; Ais an optional N-terminal amino acid sequence; B is an optionalC-terminal amino acid sequence; and n is 2, 3, 4 or 5.

If a -X- moiety has a leader peptide sequence in its wild-type form,this may be included or omitted in the hybrid protein. In someembodiments, the leader peptides will be deleted except for that of the-X- moiety located at the N-terminus of the hybrid protein i.e. theleader peptide of X₁ will be retained, but the leader peptides of X₂ . .. X_(n) will be omitted. This is equivalent to deleting all leaderpeptides and using the leader peptide of X₁ as moiety -A-.

For each n instances of [-X-L-], linker amino acid sequence -L- may bepresent or absent. For instance, when n=2 the hybrid may beNH₂—X₁-L₁-X₂-L₂-COOH, NH₂—X₁—X₂—COOH, NH₂—X₁-L₁-X₂—COOH,NH₂—X₁—X₂-L₂-COOH, etc. Linker amino acid sequence(s)-L- will typicallybe short (e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise short peptidesequences which facilitate cloning, poly-glycine linkers (i.e.comprising Gly_(n) where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), andhistidine tags (i.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10, or more).Other suitable linker amino acid sequences will be apparent to thoseskilled in the art. A useful linker is GSGGGG (SEQ ID 9), with theGly-Ser dipeptide being formed from a BamHI restriction site, thusaiding cloning and manipulation, and the (Gly)₄ tetrapeptide being atypical poly-glycine linker. If X_(n+1) is a ΔG protein and L_(n) is aglycine linker, this may be equivalent to X_(n+1) not being a ΔG proteinand L_(n) being absent.

-A- is an optional N-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leadersequences to direct protein trafficking, or short peptide sequenceswhich facilitate cloning or purification (e.g. histidine tags i.e.His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitableN-terminal amino acid sequences will be apparent to those skilled in theart. If X₁ lacks its own N-terminus methionine, -A-is preferably anoligopeptide (e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) whichprovides a N-terminus methionine.

-B- is an optional C-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includesequences to direct protein trafficking, short peptide sequences whichfacilitate cloning or purification (e.g. comprising histidine tags i.e.His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences whichenhance protein stability. Other suitable C-terminal amino acidsequences will be apparent to those skilled in the art.

Most preferably, n is 2. Two preferred proteins of this type are: X₁ isa 936 and X₂ is a 741; X₁ is a 287 and X₂ is a 953.

Two particularly preferred hybrid proteins of the invention are asfollows:

n A X₁ L₁ X₂ L₂ B [SEQ ID] 2 MA ΔG287 GSGGGG 953^((NL)) — — 7 2 M936^((NL)) GSGGGG ΔG741 — — 8

These two proteins may be used in combination with NadA (particularlywith SEQ ID 2).

936-ΔG741 hybrid can conveniently be prepared in highly pure form fromexpression in E. coli by a process comprising the steps of:homogenisation; centrifugation; cationic column chromatography; anioniccolumn chromatography; hydrophobic column chromatography; diafiltrationagainst a buffer; and filter sterilisation. Further details of theprocess are given in the examples.

Sequences

The invention provides a polypeptide having an amino acid sequenceselected from the group consisting of SEQ IDs 1 to 8. It also providespolypeptides having an amino acid sequence with sequence identity to anamino acid sequence selected from the group consisting of SEQ IDs 1 to8. As described above, the degree of sequence identity is preferablygreater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more).

The invention also provides a polypeptide comprising a fragment of a N.meningitidis NadA sequence, wherein said fragment retains the ability ofNadA to adhere to human epithelial cells. Fragments which retain aminoacids 24-87 of full-length NadA are thus preferred. Preferred fragmentslack the N-terminus leader peptide of said NadA and/or the C-terminusmembrane anchor domain of said NadA. This invention does not includewithin its scope any of the NadA fragments disclosed in the prior arte.g. in references 6 to 18. With reference to full-length NadA [17], SEQID 1 lacks the membrane anchor domain, and SEQ ID 2 lacks the leaderpeptide.

The invention also provides nucleic acid encoding such polypeptides.Furthermore, the invention provides nucleic acid which can hybridise tothis nucleic acid, preferably under “high stringency” conditions (e.g.65° C. in a 0.1×SSC, 0.5% SDS solution).

Polypeptides of the invention can be prepared by various means (e.g.recombinant expression, purification from cell culture, chemicalsynthesis (at least in part), etc.) and in various forms (e.g. native,fusions, non-glycosylated, lipidated, etc.). They are preferablyprepared in substantially pure form (i.e. substantially free from otherN. meningitidis or host cell proteins).

Nucleic acid according to the invention can be prepared in many ways(e.g. by chemical synthesis (at least in part), from genomic or cDNAlibraries, from the organism itself, etc.) and can take various forms(e.g. single stranded, double stranded, vectors, probes, etc.). They arepreferably prepared in substantially pure form (i.e. substantially freefrom other N. meningitidis or host cell nucleic acids).

The term “nucleic acid” includes DNA and RNA, and also their analogues,such as those containing modified backbones (e.g. phosphorothioates,etc.), and also peptide nucleic acids (PNA) etc. The invention includesnucleic acid comprising sequences complementary to those described above(eg. for antisense or probing purposes).

The invention also provides a process for producing a polypeptide of theinvention, comprising the step of culturing a host cell transformed withnucleic acid of the invention under conditions which induce polypeptideexpression.

The invention provides a process for producing a polypeptide of theinvention, comprising the step of synthesising at least part of thepolypeptide by chemical means.

The invention provides a process for producing nucleic acid of theinvention, comprising the step of amplifying nucleic acid using aprimer-based amplification method (e.g. PCR).

The invention provides a process for producing nucleic acid of theinvention, comprising the step of synthesising at least part of thenucleic acid by chemical means.

Strains

Preferred proteins of the invention comprise an amino acid sequencefound in N. meningitidis serogroup B. Within serogroup B, preferredstrains are 2996, MC58, 95N477, and 394/98. Strain 394/98 is sometimesreferred to herein as ‘NZ’, as it is a New Zealand strain.

Protein 287 is preferably from strain 2996 or, more preferably, fromstrain 394/98.

Protein 741 is preferably from serogroup B strains MC58, 2996, 394/98,or 95N477, or from serogroup C strain 90/18311. Strain MC58 is morepreferred.

Proteins 936, 953 and NadA are preferably from strain 2996.

Strains may be indicated as a subscript e.g. 741_(MC58) is protein 741from strain MC58. Unless otherwise stated, proteins mentioned herein(e.g. with no subscript) are from N. meningitidis strain 2996, which canbe taken as a ‘reference’ strain. It will be appreciated, however, thatthe invention is not in general limited by strain. As mentioned above,general references to a protein (e.g. ‘287’, ‘919’ etc.) may be taken toinclude that protein from any strain. This will typically have sequenceidentity to 2996 of 90% or more (eg. 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more).

Where a composition includes a particular protein antigen (e.g. 741 or287), the composition can include that antigen in more than one variantform e.g. the same protein, but from more than one strain. Theseproteins may be included as tandem or separate proteins.

Where hybrid proteins are used, the individual antigens within thehybrid (i.e. individual -X-moieties) may be from one or more strains.Where n=2, for instance, X₂ may be from the same strain as X₁ or from adifferent strain. Where n=3, the strains might be (i) X₁=X₂=X₃ (ii)X₁=X₂≠X₃ (iii) X₁≠X₂=X₃ (iv) X₁≠X₂≠X₃ or (v) X₁=X₃≠X₂, etc.

Hypervirulent Lineages and Bactericidal Antibody Responses

In general, compositions of the invention are able to induce serumbactericidal antibody responses after being administered to a subject.These responses are conveniently measured in mice and are a standardindicator of vaccine efficacy [e.g. see end-note 14 of reference 13].Serum bactericidal activity (SBA) measures bacterial killing mediated bycomplement, and can be assayed using human or baby rabbit complement.WHO standards require a vaccine to induce at least a 4-fold rise in SBAin more than 90% of recipients.

Rather than offering narrow protection, compositions of the inventioncan induce bactericidal antibody responses against more than onehypervirulent lineage of serogroup B. In particular, they can inducebactericidal responses against two or three of the following threehypervirulent lineages: (i) cluster A4; (ii) ET5 complex; and (iii)lineage 3. They may additionally induce bactericidal antibody responsesagainst one or more of hypervirulent lineages subgroup I, subgroup III,subgroup IV-1 or ET-37 complex, and against other lineages e.g.hyperinvasive lineages.

This does not necessarily mean that the composition can inducebactericidal antibodies against each and every strain of serogroup Bmeningococcus within these hypervirulent lineages e.g. rather, for anygiven group of four of more strains of serogroup B meningococcus withina particular hypervirulent lineage, the antibodies induced by thecomposition are bactericidal against at least 50% (e.g. 60%, 70%, 80%,90% or more) of the group. Preferred groups of strains will includestrains isolated in at least four of the following countries: GB, AU,CA, NO, IT, US, NZ, NL, BR, and CU. The serum preferably has abactericidal titre of at least 1024 (e.g. 2¹⁰, 2¹¹, 2¹², 2¹³, 2¹⁴, 2¹⁵,2¹⁶, 2¹⁷, 2¹⁸ or higher, preferably at least 2¹⁴) i.e. the serum is ableto kill at least 50% of test bacteria of a particular strain whendiluted 1/1024, as described in reference 13.

Preferred compositions can induce bactericidal responses against thefollowing strains of serogroup B meningococcus: (i) from cluster A4,strain 961-5945 (B:2b:P1.21, 16) and/or strain G2136 (B:-); (ii) fromET-5 complex, strain MC58 (B:15:P1.7, 16b) and/or strain 44/76(B:15:P1.7, 16); (iii) from lineage 3, strain 394/98 (B:4:P1.4) and/orstrain BZ198 (B:NT:-). More preferred compositions can inducebactericidal responses against strains 961-5945, 44/76 and 394/98.

Strains 961-5945 and G2136 are both Neisseria MLST reference strains[ids 638 & 1002 in ref. 22]. Strain MC58 is widely available (e.g. ATCCBAA-335) and was the strain sequenced in reference 6. Strain 44/76 hasbeen widely used and characterised (e.g. ref. 23) and is one of theNeisseria MLST reference strains [id 237 in ref. 22; row 32 of Table 2in ref. 1]. Strain 394/98 was originally isolated in New Zealand in1998, and there have been several published studies using this strain(e.g. refs. 24 & 25). Strain BZ198 is another MLST reference strain [id409 in ref. 22; row 41 of Table 2 in ref. 1].

The composition may additionally induce a bactericidal response againstserogroup W135 strain LNP17592 (W135:2a:P1.5, 2), from ET-37 complex.This is a Haji strain isolated in France in 2000.

Heterologous Host

Whilst expression of the proteins of the invention may take place inNeisseria, the present invention preferably utilises a heterologoushost. The heterologous host may be prokaryotic (e.g. a bacterium) oreukaryotic. It is preferably E. coli, but other suitable hosts includeBacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonellatyphimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (e.g.M. tuberculosis), yeast, etc.

Thus the invention provides a composition which, after administration toa subject, is able to induce an antibody response in that subject,wherein the antibody response is bactericidal against two or more (e.g.2 or 3) of hypervirulent lineages A4, ET-5 and lineage 3 of N.meningitidis serogroup B, and wherein the immunogens in the compositionwhich give rise to the antibody response are obtained by recombinantexpression in a non-Neisserial host. Thus the immunogens in thecompositions of the invention are preferably recombinant immunogens.Compositions which do not include OMV preparations may thus bepreferred.

Immunogenic Compositions and Medicaments

Compositions of the invention are immunogenic, and are more preferablyvaccine compositions. Vaccines according to the invention may either beprophylactic (i.e. to prevent infection) or therapeutic (i.e. to treatinfection), but will typically be prophylactic.

The pH of the composition is preferably between 6 and 8, preferablyabout 7. Stable pH may be maintained by the use of a buffer. Where acomposition comprises an aluminium hydroxide salt, it is preferred touse a histidine buffer [26]. The composition may be sterile and/orpyrogen-free. Compositions of the invention may be isotonic with respectto humans.

Compositions may be presented in vials, or they may be presented inready-filled syringes. The syringes may be supplied with or withoutneedles. A syringe will include a single dose of the composition,whereas a vial may include a single dose or multiple doses. Injectablecompositions will usually be liquid solutions or suspensions.Alternatively, they may be presented in solid form (e.g. freeze-dried)for solution or suspension in liquid vehicles prior to injection.

Compositions of the invention may be packaged in unit dose form or inmultiple dose form. For multiple dose forms, vials are preferred topre-filled syringes. Effective dosage volumes can be routinelyestablished, but a typical human dose of the composition for injectionhas a volume of 0.5 ml.

Where a composition of the invention is to be prepared extemporaneouslyprior to use (e.g. where a component is presented in lyophilised form)and is presented as a kit, the kit may comprise two vials, or it maycomprise one ready-filled syringe and one vial, with the contents of thesyringe being used to reactivate the contents of the vial prior toinjection.

The invention also provides a composition of the invention for use as amedicament. The medicament is preferably able to raise an immuneresponse in a mammal (i.e. it is an immunogenic composition) and is morepreferably a vaccine.

The invention also provides the use of a composition of the invention inthe manufacture of a medicament for raising an immune response in amammal. It also provides the use of a ‘NadA’ protein, a ‘741’ protein, a‘936’ protein, a ‘953’ protein, and a ‘287’ protein (and other optionalantigens) in the manufacture of a medicament for raising an immuneresponse in a mammal. The medicament is preferably a vaccine.

The invention also provides a method for raising an immune response in amammal comprising the step of administering an effective amount of acomposition of the invention. The immune response is preferablyprotective and preferably involves antibodies. The method may raise abooster response.

The mammal is preferably a human. Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant); wherethe vaccine is for therapeutic use, the human is preferably an adult. Avaccine intended for children may also be administered to adults e.g. toassess safety, dosage, immunogenicity, etc.

These uses and methods are preferably for the prevention and/ortreatment of a disease caused by a Neisseria (e.g. meningitis,septicaemia, bacteremia, gonorrhoea etc.). The prevention and/ortreatment of bacterial or meningococcal meningitis is preferred.

One way of checking efficacy of therapeutic treatment involvesmonitoring Neisserial infection after administration of the compositionof the invention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses against the five basic antigensafter administration of the composition. Immunogenicity of compositionsof the invention can be determined by administering them to testsubjects (e.g. children 12-16 months age, or animal models [27]) andthen determining standard parameters including serum bactericidalantibodies (SBA) and ELISA titres (GMT) of total and high-avidityanti-capsule IgG. These immune responses will generally be determinedaround 4 weeks after administration of the composition, and compared tovalues determined before administration of the composition. A SBAincrease of at least 4-fold or 8-fold is preferred. Where more than onedose of the composition is administered, more than onepost-administration determination may be made.

Preferred compositions of the invention can confer an antibody titre ina patient that is superior to the criterion for seroprotection for eachantigenic component for an acceptable percentage of human subjects.Antigens with an associated antibody titre above which a host isconsidered to be seroconverted against the antigen are well known, andsuch titres are published by organisations such as WHO. Preferably morethan 80% of a statistically significant sample of subjects isseroconverted, more preferably more than 90%, still more preferably morethan 93% and most preferably 96-100%.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, intranasal, ocular, aural, pulmonary or othermucosal administration. Intramuscular administration to the thigh or theupper arm is preferred. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dose is 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. A primary dose schedule maybe followed by a booster dose schedule. Suitable timing between primingdoses (e.g. between 4-16 weeks), and between priming and boosting, canbe routinely determined.

Neisserial infections affect various areas of the body and so thecompositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g. a lyophilised composition). The composition may beprepared for topical administration e.g. as an ointment, cream orpowder. The composition be prepared for oral administration e.g. as atablet or capsule, or as a syrup (optionally flavoured). The compositionmay be prepared for pulmonary administration e.g. as an inhaler, using afine powder or a spray. The composition may be prepared as a suppositoryor pessary. The composition may be prepared for nasal, aural or ocularadministration e.g. as spray, drops, gel or powder [e.g. refs 28 & 29].Success with nasal administration of pneumococcal saccharides [30,31],pneumococcal polypeptides [32], Hib saccharides [33], MenC saccharides[34], and mixtures of Hib and MenC saccharide conjugates [35] has beenreported.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of antigen(s), as well as any other components, asneeded. By ‘immunologically effective amount’, it is meant that theadministration of that amount to an individual, either in a single doseor as part of a series, is effective for treatment or prevention. Thisamount varies depending upon the health and physical condition of theindividual to be treated, age, the taxonomic group of individual to betreated (e.g. non-human primate, primate, etc.), the capacity of theindividual's immune system to synthesise antibodies, the degree ofprotection desired, the formulation of the vaccine, the treatingdoctor's assessment of the medical situation, and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials, and a typicalquantity of each meningococcal saccharide antigen per dose is between 1μg and 20 μg e.g. about 1 μg, about 2.5 μg, about 4 μg, about 5 μg, orabout 10 μg (expressed as saccharide).

Further Non-Antigen Components of the Composition

The composition of the invention will typically, in addition to thecomponents mentioned above, comprise one or more ‘pharmaceuticallyacceptable carriers’, which include any carrier that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolised macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,sucrose [36], trehalose [37], lactose, and lipid aggregates (such as oildroplets or liposomes). Such carriers are well known to those ofordinary skill in the art. The vaccines may also contain diluents, suchas water, saline, glycerol, etc. Additionally, auxiliary substances,such as wetting or emulsifying agents, pH buffering substances, and thelike, may be present. Sterile pyrogen-free, phosphate-bufferedphysiologic saline is a typical carrier. A thorough discussion ofpharmaceutically acceptable excipients is available in reference 38.

Compositions of the invention may include an antimicrobial, particularlywhen packaged in multiple dose format.

Compositions of the invention may comprise detergent e.g. a Tween(polysorbate), such as Tween 80. Detergents are generally present at lowlevels e.g. <0.01%.

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical.

Compositions of the invention will generally include a buffer. Aphosphate buffer is typical.

Compositions of the invention may comprise a sugar alcohol (e.g.mannitol) or a disaccharide (e.g. sucrose or trehalose) e.g. at around15-30 mg/ml (e.g. 25 mg/ml), particularly if they are to be lyophilisedor if they include material which has been reconstituted fromlyophilised material. The pH of a composition for lyophilisation may beadjusted to around 6.1 prior to lyophilisation.

Vaccines of the invention may be administered in conjunction with otherimmunoregulatory agents. In particular, compositions will usuallyinclude an adjuvant. Adjuvants which may be used in compositions of theinvention include, but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. [e.g. see chapters 8 & 9 of ref. 39], or mixtures ofdifferent mineral compounds, with the compounds taking any suitable form(e.g. gel, crystalline, amorphous, etc.), and with adsorption beingpreferred. The mineral containing compositions may also be formulated asa particle of metal salt [40].

Aluminium phosphates are particularly preferred, particularly incompositions which include a H. influenzae saccharide antigen, and atypical adjuvant is amorphous aluminium hydroxyphosphate with PO₄/Almolar ratio between 0.84 and 0.92, included at 0.6 mg Al³⁺/ml.Adsorption with a low dose of aluminium phosphate may be used e.g.between 50 and 100 μg Al³⁺per conjugate per dose. Where there is morethan one conjugate in a composition, not all conjugates need to beadsorbed.

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 [Chapter 10 of ref. 39;see also ref. 41] (5% Squalene, 0.5% Tween 80, and 0:5% Span 85,formulated into submicron particles using a microfluidizer). CompleteFreund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may alsobe used.

C. Saponin Formulations [Chapter 22 of Ref 39]

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree have been widely studied as adjuvants.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS 17, QS 18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. A method of production of QS21 is disclosed in ref. 42.Saponin formulations may also comprise a sterol, such as cholesterol[43].

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexs (ISCOMs) [chapter 23 of ref.39]. ISCOMs typically also include a phospholipid such asphosphatidylethanolamine or phosphatidylcholine. Any known saponin canbe used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA,QHA & QHC. ISCOMs are further described in refs. 43-45. Optionally, theISCOMS may be devoid of additional detergent [46].

A review of the development of saponin based adjuvants can be found inrefs. 47 & 48.

D. Virosomes and Virus-Like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin refs. 49-54. Virosomes are discussed further in, for example, ref. 55

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred“small particle” form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in ref. 56. Such “small particles” of 3dMPL are small enoughto be sterile filtered through a 0.22 μm membrane [56]. Other non-toxicLPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [57,58].

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 59 & 60.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 61, 62 and 63 disclose possible analogsubstitutions e.g. replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 64-69.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT [70]. The CpG sequence may be specific for inducing a Th1 immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inrefs. 71-73. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, refs. 70 & 74-76.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref. 77 and as parenteraladjuvants in ref. 78. The toxin or toxoid is preferably in the form of aholotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant is a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivatives thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 79-86. Numerical reference for aminoacid substitutions is preferably based on the alignments of the A and Bsubunits of ADP-ribosylating toxins set forth in ref. 87, specificallyincorporated herein by reference in its entirety.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12 [88], etc.) [89], interferons (e.g. interferon-γ),macrophage colony stimulating factor, and tumor necrosis factor.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres [90] or mucoadhesives such as cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention [91].

H. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

I. Liposomes (Chapters 13 & 14 of Ref 39)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 92-94.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters [95]. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol [96] as well as polyoxyethylene alkyl ethers or estersurfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol [97]. Preferred polyoxyethylene ethersare selected from the following group: polyoxyethylene-9-lauryl ether(laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steorylether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether,and polyoxyethylene-23-lauryl ether.

K. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in refs. 98 and 99.

L. Muramyl peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

M. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues (e,g. “Resiquimod 3M”),described further in refs. 100 and 101.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention: (1) a saponin and anoil-in-water emulsion [102]; (2) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL) [103]; (3) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL)+a cholesterol; (4) a saponin (e.g.QS21)+3dMPL+IL-12 (optionally+a sterol) [104]; (5) combinations of 3dMPLwith, for example, QS21 and/or oil-in-water emulsions [105]; (6) SAF,containing 10% squalane, 0.4% Tween 80™, 5% pluronic-block polymer L121,and thr-MDP, either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion. (7) Ribi™ adjuvant system(RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and oneor more bacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); and (8) one or more mineralsalts (such as an aluminum salt)+a non-toxic derivative of LPS (such as3dMPL).

Other substances that act as immunostimulating agents are disclosed inchapter 7 of ref. 39.

The use of an aluminium hydroxide or aluminium phosphate adjuvant isparticularly preferred, and antigens are generally adsorbed to thesesalts. Aluminium hydroxide is preferably avoided as an adjuvant if thecomposition includes a Hib antigen. Where an aluminium phosphate it usedand desired not to adsorb an antigen to the adjuvant, this is favouredby including free phosphate ions in solution (e.g. by the use of aphosphate buffer). Prevention of adsorption can also be achieved byselecting the correct pH during antigen/adjuvant mixing, an adjuvantwith an appropriate point of zero charge, and an appropriate order ofmixing for different antigens in a composition [106].

Calcium phosphate is another preferred adjuvant.

Further Antigens

Compositions of the invention contain five basic meningococcal proteinantigens. They may also include further antigens, although it maycontain no meningococcal protein antigens other than the five basicantigens. Further antigens for inclusion may be, for example:

-   -   a saccharide antigen from Haemophilus influenzae B.    -   a saccharide antigen from N. meningitidis serogroup A, C, W135        and/or Y, such as the oligosaccharide disclosed in ref. 107 from        serogroup C or the oligosaccharides of ref. 108.    -   a saccharide antigen from Streptococcus pneumoniae [e.g. 155,        156 157].    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 109, 110].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 110, 111].    -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        3 of ref. 112] e.g. the CRM₁₉₇ mutant [e.g. 113].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of        ref. 112].    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3 [e.g. refs. 114 & 115]. Cellular pertussis        antigen may be used.    -   an outer-membrane vesicle (OMV) preparation from N. meningitidis        serogroup B, such as those disclosed in refs. 4, 116, 117, 118        etc.    -   polio antigen(s) [e.g. 119, 120] such as OPV or, preferably,        IPV.

The composition may comprise one or more of these further antigens.Antigens will typically be present at a concentration of at least 1μg/ml each. In general, the concentration of any given antigen will besufficient to elicit an immune response against that antigen. It ispreferred that the protective efficacy of individual saccharide antigensis not removed by combining them, although actual immunogenicity (e.g.ELISA titres) may be reduced.

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens. Such DTP combinations can be used to reconstitutelyophilised conjugates.

Where a saccharide or carbohydrate antigen is used, it is preferablyconjugated to a carrier protein in order to enhance immunogenicity (seebelow).

Toxic protein antigens may be detoxified where necessary (e.g.detoxification of pertussis toxin by chemical and/or genetic means[115]).

As an alternative to using protein antigens in the composition of theinvention, nucleic acid encoding the antigen may be used [e.g. refs. 121to 129]. Protein components of the compositions of the invention maythus be replaced by nucleic acid (preferably DNA e.g. in the form of aplasmid) that encodes the protein. Similarly, compositions of theinvention may comprise proteins which mimic saccharide antigens e.g.mimotopes [130] or anti-idiotype antibodies. These may replaceindividual saccharide components, or may supplement them. As an example,the vaccine may comprise a peptide mimic of the MenC [131] or the MenA[132] capsular polysaccharide in place of the saccharide itself.

Particularly preferred compositions of the invention include one, two orthree of (a) saccharide antigens from meningococcus serogroups Y, W135,C and (optionally) A; (b) a saccharide antigen from Haemophilusinfluenzae type B; and/or (c) an antigen from Streptococcus pneumoniae.A composition comprising the serogroup B antigens and a Hib conjugate isparticularly preferred.

Meningococcus Serogroups Y. W135, C and (Optionally) A

As mentioned above, polysaccharide vaccines against serogroups A, C,W135 & Y has been known for many years. These vaccines (MENCEVAX ACWY™and MENOMUNE™) are based on the organisms' capsular polysaccharides and,although they are effective in adolescents and adults, they give a poorimmune response and short duration of protection, and they cannot beused in infants.

In contrast to the unconjugated polysaccharide antigens in thesevaccines, the recently-approved serogroup C vaccines (Menjugate™[133,107], Meningitec™ and NeisVac-C™) include conjugated saccharides.Menjugate™ and Meningitec™ have oligosaccharide antigens conjugated to aCRM₁₉₇ carrier, whereas NeisVac-C™ uses the complete polysaccharide(de-O-acetylated) conjugated to a tetanus toxoid carrier.

Compositions of the present invention preferably include capsularsaccharide antigens from one or more of meningococcus serogroups Y,W135, C and (optionally) A, wherein the antigens are conjugated tocarrier protein(s) and/or are oligosaccharides.

A typical quantity of each meningococcal saccharide antigen per dose isbetween 1 μg and 20 μg e.g. about 1 μg, about 2.5 μg, about 4 μg, about5 μg, or about 10 μg (expressed as saccharide).

Where a mixture comprises capsular saccharides from both serogroups Aand C, the ratio (w/w) of MenA saccharide:MenC saccharide may be greaterthan 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 or higher). Where a mixturecomprises capsular saccharides from serogroup Y and one or both ofserogroups C and W135, the ratio (w/w) of MenY saccharide:MenW135saccharide may be greater than 1 (e.g. 2:1, 3:1, 4:1, 5:1, 10:1 orhigher) and/or that the ratio (w/w) of MenY saccharide:MenC saccharidemay be less than 1 (e.g. 1:2, 1:3, 1:4, 1:5, or lower). Preferred ratios(w/w) for saccharides from serogroups A:C:W135:Y are: 1:1:1:1; 1:1:1:2;2:1:1:1; 4:2:1:1; 8:4:2:1; 4:2:1:2; 8:4:1:2; 4:2:2:1; 2:2:1:1; 4:4:2:1;2:2:1:2; 4:4:1:2; and 2:2:2:1. Preferred ratios (w/w) for saccharidesfrom serogroups C:W135:Y are: 1:1:1; 1:1:2; 1:1:1; 2:1:1; 4:2:1; 2:1:2;4:1:2; 2:2:1; and 2:1:1. Using a substantially equal mass of eachsaccharide is preferred.

Capsular saccharides will generally be used in the form ofoligosaccharides. These are conveniently formed by fragmentation ofpurified capsular polysaccharide (e.g. by hydrolysis), which willusually be followed by purification of the fragments of the desiredsize.

Fragmentation of polysaccharides is preferably performed to give a finalaverage degree of polymerisation (DP) in the oligosaccharide of lessthan 30 (e.g. between 10 and 20, preferably around 10 for serogroup A;between 15 and 25 for serogroups W135 and Y, preferably around 15-20;between 12 and 22 for serogroup C; etc.). DP can conveniently bemeasured by ion exchange chromatography or by colorimetric assays [134].

If hydrolysis is performed, the hydrolysate will generally be sized inorder to remove short-length oligosaccharides [135]. This can beachieved in various ways, such as ultrafiltration followed byion-exchange chromatography. Oligosaccharides with a degree ofpolymerisation of less than or equal to about 6 are preferably removedfor serogroup A, and those less than around 4 are preferably removed forserogroups W135 and Y.

Preferred MenC saccharide antigens are disclosed in reference 133, asused in Menjugate™.

The saccharide antigen may be chemically modified. This is particularlyuseful for reducing hydrolysis for serogroup A [136; see below].De-O-acetylation of meningococcal saccharides can be performed. Foroligosaccharides, modification may take place before or afterdepolymerisation.

Where a composition of the invention includes a MenA saccharide antigen,the antigen is preferably a modified saccharide in which one or more ofthe hydroxyl groups on the native saccharide has/have been replaced by ablocking group [136]. This modification improves resistance tohydrolysis, and means that the serogroup A antigen can be stored andused in a liquid formulation rather than requiring lyophilisation.

The number of monosaccharide units having blocking groups can vary. Forexample, all or substantially all the monosaccharide units may haveblocking groups. Alternatively, at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80% or 90% of the monosaccharide units may have blocking groups. Atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 monosaccharide units mayhave blocking groups.

Likewise, the number of blocking groups on a monosaccharide unit mayvary. For example, the number of blocking groups on a monosaccharideunit may be 1 or 2. The blocking group will generally be at the 4position and/or 3-position of the monosaccharide units.

The terminal monosaccharide unit may or may not have a blocking groupinstead of its native hydroxyl. It is preferred to retain a freeanomeric hydroxyl group on a terminal monosaccharide unit in order toprovide a handle for further reactions (e.g. conjugation). Anomerichydroxyl groups can be converted to amino groups (—NH₂ or —NH-E, where Eis a nitrogen protecting group) by reductive amination (using, forexample, NaBH₃CN/NH₄Cl), and can then be regenerated after otherhydroxyl groups have been converted to blocking groups.

Blocking groups to replace hydroxyl groups may be directly accessiblevia a derivatizing reaction of the hydroxyl group i.e. by replacing thehydrogen atom of the hydroxyl group with another group. Suitablederivatives of hydroxyl groups which act as blocking groups are, forexample, carbamates, sulfonates, carbonates, esters, ethers (e.g. silylethers or alkyl ethers) and acetals. Some specific examples of suchblocking groups are allyl, Aloc, benzyl, BOM, t-butyl, trityl, TBS,TBDPS, TES, TMS, TIPS, PMB, MEM, MOM, MTM, THP, etc. Other blockinggroups that are not directly accessible and which completely replace thehydroxyl group include C₁₋₁₂ alkyl, C₃₋₁₂ alkyl, C₅₋₁₂ aryl, C₅₋₁₂aryl-C₁₋₆alkyl, NR¹R² (R¹ and R² are defined in the followingparagraph), H, F, Cl, Br, CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃, CCl₃, etc.Preferred blocking groups are electron-withdrawing groups.

Preferred blocking groups are of the formula: —O—X—Y or —OR³ wherein: Xis C(O), S(O) or SO₂; Y is C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy, C₃₋₁₂ cycloalkyl,C₅₋₁₂ aryl or C₅₋₁₂ aryl-C₁₋₆ alkyl, each of which may optionally besubstituted with 1, 2 or 3 groups independently selected from F, Cl, Br,CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃ or CCl₃; or Y is NR¹R²; R¹ and R² areindependently selected from H, C₁₋₁₂ alkyl, C₃₋₁₂ cycloalkyl, C₅₋₁₂aryl, C₅₋₁₂ aryl-C₁₋₆ alkyl; or R¹ and R² may be joined to form a C₃₋₁₂saturated heterocyclic group; R³ is C₁₋₁₂ alkyl or C₃₋₁₂ cycloalkyl,each of which may optionally be substituted with 1, 2 or 3 groupsindependently selected from F, Cl, Br, CO₂(C₁₋₆ alkyl), CN, CF₃ or CCl₃;or R³ is C₅₋₁₂ aryl or C₅₋₁₂ aryl-C₁₋₆ alkyl, each of which mayoptionally be substituted with 1, 2, 3, 4 or 5 groups selected from F,Cl, Br, CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃ or CCl₃. When R³ is C₁₋₁₂ alkylor C₃₋₁₂ cycloalkyl, it is typically substituted with 1, 2 or 3 groupsas defined above. When R¹ and R² are joined to form a C₃₋₁₂ saturatedheterocyclic group, it is meant that R¹ and R² together with thenitrogen atom form a saturated heterocyclic group containing any numberof carbon atoms between 3 and 12 (e.g. C₃, C₄, C_(s), C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂). The heterocyclic group may contain 1 or 2 heteroatoms(such as N, O or S) other than the nitrogen atom. Examples of C₃₋₁₂saturated heterocyclic groups are pyrrolidinyl, piperidinyl,morpholinyl, piperazinyl, imidazolidinyl, azetidinyl and aziridinyl.

Blocking groups —O—X—Y and —OR³ can be prepared from —OH groups bystandard derivatizing procedures, such as reaction of the hydroxyl groupwith an acyl halide, alkyl halide, sulfonyl halide, etc. Hence, theoxygen atom in —O—X—Y is preferably the oxygen atom of the hydroxylgroup, while the —X—Y group in —O—X—Y preferably replaces the hydrogenatom of the hydroxyl group.

Alternatively, the blocking groups may be accessible via a substitutionreaction, such as a Mitsonobu-type substitution. These and other methodsof preparing blocking groups from hydroxyl groups are well known.

More preferably, the blocking group is —OC(O)CF_(3 [)137], or acarbamate group —OC(O)NR¹R², where R¹ and R² are independently selectedfrom C₁₋₆ alkyl. More preferably, R¹ and R² are both methyl i.e. theblocking group is —OC(O)NMe₂. Carbamate blocking groups have astabilizing effect on the glycosidic bond and may be prepared under mildconditions.

Preferred modified MenA saccharides contain n monosaccharide units,where at least h % of the monosaccharide units do not have —OH groups atboth of positions 3 and 4. The value of h is 24 or more (e.g. 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98,99 or 100) and is preferably 50 or more. The absent —OH groups arepreferably blocking groups as defined above.

Other preferred modified MenA saccharides comprise monosaccharide units,wherein at least s of the monosaccharide units do not have —OH at the 3position and do not have —OH at the 4 position. The value of s is atleast 1 (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90). The absent —OH groups are preferably blocking groups asdefined above.

Suitable modified MenA saccharides for use with the invention have theformula:

wherein

-   -   n is an integer from 1 to 100 (preferably an integer from 15 to        25);    -   T is of the formula (A) or (B):

-   -   each Z group is independently selected from OH or a blocking        group as defined above; and    -   each Q group is independently selected from OH or a blocking        group as defined above;    -   Y is selected from OH or a blocking group as defined above;    -   E is H or a nitrogen protecting group;        and wherein more than about 7% (e.g. 8%, 9%, 10% or more) of the        Q groups are blocking groups.

Each of the n+2 Z groups may be the same or different from each other.Likewise, each of the n+2 Q groups may be the same or different fromeach other. All the Z groups may be OH. Alternatively, at least 10%, 20,30%, 40%, 50% or 60% of the Z groups may be OAc. Preferably, about 70%of the Z groups are OAc, with the remainder of the Z groups being OH orblocking groups as defined above. At least about 7% of Q groups areblocking groups. Preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or even 100% of the Q groups are blocking groups.

Preferred compositions of the invention can be stored for 28 days at 37°C. and, after that period, less than f % of the initial total amount ofconjugated MenA saccharide will be unconjugated, where f is 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or lower.

Meningococcal capsular polysaccharides are typically prepared by aprocess comprising the steps of polysaccharide precipitation (e.g. usinga cationic detergent), ethanol fractionation, cold phenol extraction (toremove protein) and ultracentrifugation (to remove LPS) [e.g. ref. 138].A more preferred process [108], however, involves polysaccharideprecipitation followed by solubilisation of the precipitatedpolysaccharide using a lower alcohol. Precipitation can be achievedusing a cationic detergent such as tetrabutylammonium andcetyltrimethylammonium salts (e.g. the bromide salts), or hexadimethrinebromide and myristyltrimethylammonium salts. Cetyltrimethylammoniumbromide (‘CTAB’) is particularly preferred [139]. Solubilisation of theprecipitated material can be achieved using a lower alcohol such asmethanol, propan-1-ol, propan-2-ol, butan-1-ol, butan-2-ol,2-methyl-propan-1-ol, 2-methyl-propan-2-ol, diols, etc., but ethanol isparticularly suitable for solubilising CTAB-polysaccharide complexes.Ethanol is preferably added to the precipitated polysaccharide to give afinal concentration (based on total content of ethanol and water) ofbetween 50% and 95%.

After re-solubilisation, the polysaccharide may be further treated toremove contaminants. This is particularly important in situations whereeven minor contamination is not acceptable (e.g. for human vaccineproduction). This will typically involve one or more steps of filtratione.g. depth filtration, filtration through activated carbon may be used,size filtration and/or ultrafiltration. Once filtered to removecontaminants, the polysaccharide may be precipitated for furthertreatment and/or processing. This can be conveniently achieved byexchanging cations (e.g. by the addition of calcium or sodium salts).

As an alternative to purification, capsular saccharides of the presentinvention may be obtained by total or partial synthesis e.g. Hibsynthesis is disclosed in ref. 140, and MenA synthesis in ref. 141.

Compositions of the invention comprise capsular saccharides from atleast two serogroups of N. meningitidis. The saccharides are preferablyprepared separately (including any fragmentation, conjugation,modification, etc.) and then admixed to give a composition of theinvention.

Where the composition comprises capsular saccharide from serogroup A,however, it is preferred that the serogroup A saccharide is not combinedwith the other saccharide(s) until shortly before use, in order tominimise the potential for hydrolysis. This can conveniently be achievedby having the serogroup A component (typically together with appropriateexcipients) in lyophilised form and the other serogroup component(s) inliquid form (also with appropriate excipients), with the liquidcomponents being used to reconstitute the lyophilised MenA componentwhen ready for use. Where an aluminium salt adjuvant is used, it ispreferred to include the adjuvant in the vial containing the with theliquid vaccine, and to lyophilise the MenA component without adjuvant.

A composition of the invention may thus be prepared from a kitcomprising: (a) capsular saccharide from N. meningitidis serogroup A, inlyophilised form; and (b) the further antigens from the composition, inliquid form. The invention also provides a method for preparing acomposition of the invention, comprising mixing a lyophilised capsularsaccharide from N. meningitidis serogroup A with the further antigens,wherein said further antigens are in liquid form.

The invention also provides a kit comprising: (a) a first containercontaining capsular saccharides from two or more of N. meningitidisserogroups C, W135 and Y, all in lyophilised form; and (b) a secondcontainer containing in liquid form (i) a composition which, afteradministration to a subject, is able to induce an antibody response inthat subject, wherein the antibody response is bactericidal against twoor more (e.g. 2 or 3) of hypervirulent lineages A4, ET-5 and lineage 3of N. meningitidis serogroup B, (ii) capsular saccharides from none orone of N. meningitidis serogroups C, W135 and Y, and optionally (iii)further antigens (see below) that do not include meningococcal capsularsaccharides, wherein, reconstitution of the contents of container (a) bythe contents of container (b) provides a composition of the invention.

Within each dose, the amount of an individual saccharide antigen willgenerally be between 1-50 μg (measured as mass of saccharide), withabout 2.5 μg, 5 μg or 10 μg of each being preferred. With A:C:W135:Yweight ratios of 1:1:1:1; 1:1:1:2; 2:1:1:1; 4:2:1:1; 8:4:2:1; 4:2:1:2;8:4:1:2; 4:2:2:1; 2:2:1:1; 4:4:2:1; 2:2:1:2; 4:4:1:2; and 2:2:2:1,therefore, the amount represented by the FIG. 1 is preferably about 2.5μg, 5 μg or 10 μg. For a 1:1:1:1 ratio A:C:W:Y composition and a 10 μgper saccharide, therefore, 40 μg saccharide is administered per dose.Preferred compositions have about the following μg saccharide per dose:

A 10 0 0 0 10 5 2.5 C 10 10 5 2.5 5 5 2.5 W135 10 10 5 2.5 5 5 2.5 Y 1010 5 2.5 5 5 2.5

Preferred compositions of the invention comprise less than 50 μgmeningococcal saccharide per dose. Other preferred compositions comprise≦40 μg meningococcal saccharide per dose. Other preferred compositionscomprise ≦30 μg meningococcal saccharide per dose. Other preferredcompositions comprise ≦25 μg meningococcal saccharide per dose. Otherpreferred compositions comprise ≦20 μg meningococcal saccharide perdose. Other preferred compositions comprise ≦10 μg meningococcalsaccharide per dose but, ideally, compositions of the invention compriseat least 10 μg meningococcal saccharide per dose.

The Menjugate™ and NeisVac™ MenC conjugates use a hydroxide adjuvant,whereas Meningitec™ uses a phosphate. It is possible in compositions ofthe invention to adsorb some antigens to an aluminium hydroxide but tohave other antigens in association with an aluminium phosphate. Fortetravalent serogroup combinations, for example, the followingpermutations are available:

Serogroup Aluminium salt (H = a hydroxide; P = a phosphate) A P H P H HH P P P H H H P P P H C P H H P H H P H H P P H P H P P W135 P H H H P HH P H H P P P P H P Y P H H H H P H H P P H P H P P P

For trivalent N. meningitidis serogroup combinations, the followingpermutations are available:

Serogroup Aluminium salt (H = a hydroxide; P = a phosphate) C P H H H PP P H W135 P H H P H P H P Y P H P H H H P PHaemophilus influenzae Type B

Where the composition includes a H. influenzae type B antigen, it willtypically be a Hib capsular saccharide antigen. Saccharide antigens fromH. influenzae b are well known.

Advantageously, the Hib saccharide is covalently conjugated to a carrierprotein, in order to enhance its immunogenicity, especially in children.The preparation of polysaccharide conjugates in general, and of the Hibcapsular polysaccharide in particular, is well documented [e.g.references 142 to 150 etc.]. The invention may use any suitable Hibconjugate. Suitable carrier proteins are described below, and preferredcarriers for Hib saccharides are CRM₁₉₇ (‘HbOC’), tetanus toxoid(‘PRP-T’) and the outer membrane complex of N. meningitidis (‘PRP-OMP’).

The saccharide moiety of the conjugate may be a polysaccharide (e.g.full-length polyribosylribitol phosphate (PRP)), but it is preferred tohydrolyse polysaccharides to form oligosaccharides (e.g. MW from ˜1 to˜5 kDa).

A preferred conjugate comprises a Hib oligosaccharide covalently linkedto CRM₁₉₇ via an adipic acid linker [151, 152]. Tetanus toxoid is also apreferred carrier.

Administration of the Hib antigen preferably results in an anti-PRPantibody concentration of ≧0.15 μg/ml, and more preferably ≧1 μg/ml.

Compositions of the invention may comprise more than one Hib antigen.

Where a composition includes a Hib saccharide antigen, it is preferredthat it does not also include an aluminium hydroxide adjuvant. If thecomposition includes an aluminium phosphate adjuvant then the Hibantigen may be adsorbed to the adjuvant [153] or it may be non-adsorbed[154].

Hib antigens may be lyophilised e.g. together with meningococcalantigens.

Streptococcus pneumoniae

Where the composition includes a S. pneumoniae antigen, it willtypically be a capsular saccharide antigen which is preferablyconjugated to a carrier protein [e.g. refs. 155 to 157]. It is preferredto include saccharides from more than one serotype of S. pneumoniae. Forexample, mixtures of polysaccharides from 23 different serotype arewidely used, as are conjugate vaccines with polysaccharides from between5 and 11 different serotypes [158]. For example, PrevNar™ [159] containsantigens from seven serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) witheach saccharide individually conjugated to CRM₁₉₇ by reductiveamination, with 2 μg of each saccharide per 0.5 ml dose (4 μg ofserotype 6B), and with conjugates adsorbed on an aluminium phosphateadjuvant. Compositions of the invention preferably include at leastserotypes 6B, 14, 19F and 23F. Conjugates may be adsorbed onto analuminium phosphate.

As an alternative to using saccharide antigens from pneumococcus, thecomposition may include one or more polypeptide antigens. Genomesequences for several strains of pneumococcus are available [160,161]and can be subjected to reverse vaccinology [162-165] to identifysuitable polypeptide antigens [166,167]. For example, the compositionmay include one or more of the following antigens: PhtA, PhtD, PhtB,PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp130, asdefined in reference 168. The composition may include more than one(e.g. 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13 or 14) of these antigens.

In some embodiments, the composition may include both saccharide andpolypeptide antigens from pneumococcus. These may be used in simpleadmixture, or the pneumococcal saccharide antigen may be conjugated to apneumococcal protein. Suitable carrier proteins for such embodimentsinclude the antigens listed in the previous paragraph [168].

Pneumococcal antigens may be lyophilised e.g. together withmeningococcal and/or Hib antigens.

Covalent Conjugation

Capsular saccharides in compositions of the invention will usually beconjugated to carrier protein(s). In general, conjugation enhances theimmunogenicity of saccharides as it converts them from T-independentantigens to T-dependent antigens, thus allowing priming forimmunological memory. Conjugation is particularly useful for pediatricvaccines and is a well known technique [e.g. reviewed in refs. 169 and142-150].

Preferred carrier proteins are bacterial toxins or toxoids, such asdiphtheria toxoid or tetanus toxoid. The CRM₁₉₇ diphtheria toxoid[170-172] is particularly preferred. Other suitable carrier proteinsinclude the N. meningitidis outer membrane protein [173], syntheticpeptides [174,175], heat shock proteins [176,177], pertussis proteins[178,179], cytokines [180], lymphokines [180], hormones [180], growthfactors [180], artificial proteins comprising multiple human CD4⁺ T cellepitopes from various pathogen-derived antigens [181], protein D from H.influenzae [182,183], pneumococcal surface protein PspA [184],iron-uptake proteins [185], toxin A or B from C. difficile [186], etc.Preferred carriers are diphtheria toxoid, tetanus toxoid, H. influenzaeprotein D, and CRM₁₉₇.

Within a composition of the invention, it is possible to use more thanone carrier protein e.g. to reduce the risk of carrier suppression. Thusdifferent carrier proteins can be used for different serogroups e.g.serogroup A saccharides might be conjugated to CRM₁₉₇ while serogroup Csaccharides might be conjugated to tetanus toxoid. It is also possibleto use more than one carrier protein for a particular saccharide antigene.g. serogroup A saccharides might be in two groups, with someconjugated to CRM₁₉₇ and others conjugated to tetanus toxoid. Ingeneral, however, it is preferred to use the same carrier protein forall saccharides.

A single carrier protein might carry more than one saccharide antigen[187]. For example, a single carrier protein might have conjugated to itsaccharides from serogroups A and C. To achieve this goal, saccharidescan be mixed prior to the conjugation reaction. In general, however, itis preferred to have separate conjugates for each serogroup.

Conjugates with a saccharide:protein ratio (w/w) of between 1:5 (i.e.excess protein) and 5:1 (i.e. excess saccharide) are preferred. Ratiosbetween 1:2 and 5:1 are preferred, as are ratios between 1:1.25 and1:2.5 are more preferred. Excess carrier protein may be preferred forMenA and MenC.

Conjugates may be used in conjunction with free carrier protein [188].When a given carrier protein is present in both free and conjugated formin a composition of the invention, the unconjugated form is preferablyno more than 5% of the total amount of the carrier protein in thecomposition as a whole, and more preferably present at less than 2% byweight.

Any suitable conjugation reaction can be used, with any suitable linkerwhere necessary.

The saccharide will typically be activated or functionalised prior toconjugation. Activation may involve, for example, cyanylating reagentssuch as CDAP (e.g. 1-cyano-4-dimethylamino pyridinium tetrafluoroborate[189,190, etc.]). Other suitable techniques use carbodiimides,hydrazides, active esters, norborane, p-nitrobenzoic acid,N-hydroxysuccinimide, S—NHS, EDC, TSTU; see also the introduction toreference 148).

Linkages via a linker group may be made using any known procedure, forexample, the procedures described in references 191 and 192. One type oflinkage involves reductive amination of the polysaccharide, coupling theresulting amino group with one end of an adipic acid linker group, andthen coupling a protein to the other end of the adipic acid linker group[146,193,194]. Other linkers include B-propionamido [195],nitrophenyl-ethylamine [196], haloacyl halides [197], glycosidiclinkages [198], 6-aminocaproic acid [199], ADH [200], C₄ to C₁₂ moieties[201] etc. As an alternative to using a linker, direct linkage can beused. Direct linkages to the protein may comprise oxidation of thepolysaccharide followed by reductive amination with the protein, asdescribed in, for example, references 202 and 203.

A process involving the introduction of amino groups into the saccharide(e.g. by replacing terminal ═O groups with —NH₂) followed byderivatisation with an adipic diester (e.g. adipic acidN-hydroxysuccinimido diester) and reaction with carrier protein ispreferred. Another preferred reaction uses CDAP activation with aprotein D carrier e.g. for MenA or MenC.

After conjugation, free and conjugated saccharides can be separated.There are many suitable methods, including hydrophobic chromatography,tangential ultrafiltration, diafiltration etc. [see also refs. 204 &205, etc.].

Where the composition of the invention includes a conjugatedoligosaccharide, it is preferred that oligosaccharide preparationprecedes conjugation.

Further and Alternative Serogroup B Polypeptide Antigens

The invention provides a composition which, after administration to asubject, is able to induce an antibody response in that subject, whereinthe antibody response is bactericidal against two or three ofhypervirulent lineages A4, ET-5 and lineage 3 of N. meningitidisserogroup B.

Although NadA, 741, 936, 953 and 287 are preferred antigens forachieving this broad protection, other MenB polypeptide antigens whichmay be included in compositions of the invention (optionally incombination with one or more of the five basic antigens) include thosecomprising one of the following amino acid sequences: SEQ ID NO:650 fromref. 8; SEQ ID NO:878 from ref. 8; SEQ ID NO:884 from ref. 8; SEQ IDNO:4 from ref. 9; SEQ ID NO:598 from ref. 10; SEQ ID NO:818 from ref.10; SEQ ID NO:864 from ref. 10; SEQ ID NO:866 from ref. 10; SEQ IDNO:1196 from ref. 10; SEQ ID NO:1272 from ref. 10; SEQ ID NO:1274 fromref. 10; SEQ ID NO:1640 from ref. 10; SEQ ID NO:1788 from ref. 10; SEQID NO:2288 from ref. 10; SEQ ID NO:2466 from ref. 10; SEQ ID NO:2554from ref. 10; SEQ ID NO:2576 from ref. 10; SEQ ID NO:2606 from ref. 10;SEQ ID NO:2608 from ref. 10; SEQ ID NO:2616 from ref. 10; SEQ ID NO:2668from ref. 10; SEQ ID NO:2780 from ref. 10; SEQ ID NO:2932 from ref. 10;SEQ ID NO:2958 from ref. 10; SEQ ID NO:2970 from ref. 10; SEQ ID NO:2988from ref. 10, or a polypeptide comprising an amino acid sequence which:(a) has 50% or more identity (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more)to said sequences; and/or (b) comprises a fragment of at least nconsecutive amino acids from said sequences, wherein n is 7 or more (eg.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments for (b) comprise an epitope fromthe relevant sequence. More than one (e.g. 2, 3, 4, 5, 6) of thesepolypeptides may be included.

General

The term “comprising” means “including” as well as “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x+10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to a percentage sequence identity between two amino acidsequences means that, when aligned, that percentage of amino acids arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 ofreference 206. A preferred alignment is determined by the Smith-Watermanhomology search algorithm using an affine gap search with a gap openpenalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is taught in reference 207.

The term “alkyl” refers to alkyl groups in both straight and branchedforms, The alkyl group may be interrupted with 1, 2 or 3 heteroatomsselected from —O—, —NH— or —S—. The alkyl group may also be interruptedwith 1, 2 or 3 double and/or triple bonds. However, the term “alkyl”usually refers to alkyl groups having no heteroatom interruptions ordouble or triple bond interruptions. Where reference is made to C₁₋₁₂alkyl, it is meant the alkyl group may contain any number of carbonatoms between 1 and 12 (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀,C₁₁C₁₂). Similarly, where reference is made to C₁₋₆ alkyl, it is meantthe alkyl group may contain any number of carbon atoms between 1 and 6(e.g. C₁, C₂, C₃, C₄, C₅, C₆).

The term “cycloalkyl” includes cycloalkyl, polycycloalkyl, andcycloalkenyl groups, as well as combinations of these with alkyl groups,such as cycloalkylalkyl groups. The cycloalkyl group may be interruptedwith 1, 2 or 3 heteroatoms selected from —O—, —NH— or —S—. However, theterm “cycloalkyl” usually refers to cycloalkyl groups having noheteroatom interruptions Examples of cycloalkyl groups includecyclopentyl, cyclohexyl, cyclohexenyl, cyclohexylmethyl and adamantylgroups. Where reference is made to C₃₋₁₂ cycloalkyl, it is meant thatthe cycloalkyl group may contain any number of carbon atoms between 3and 12 (e.g. C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂).

The term “aryl” refers to an aromatic group, such as phenyl or naphthyl.Where reference is made to C₅₋₁₂ aryl, it is meant that the aryl groupmay contain any number of carbon atoms between 5 and 12 (e.g. C₅, C₆,C₇, C₈, C₉, C₁₀, C₁₁, C₁₂).

The term “C₅₋₁₂aryl-C₁₋₆alkyl” refers to groups such as benzyl,phenylethyl and naphthylmethyl.

Nitrogen protecting groups include silyl groups (such as TMS, TES, TBS,TIPS), acyl derivatives (such as phthalimides, trifluoroacetamides,methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl (Boc),benzyloxycarbonyl (Z or Cbz), 9-fluorenylmethoxycarbonyl (Fmoc),2-(trimethylsilyl)ethoxy carbonyl, 2,2,2-trichloroethoxycarbonyl(Troc)), sulfonyl derivatives (such as β-trimethylsilylethanesulfonyl(SES)), sulfenyl derivatives, C₁₋₁₂ alkyl, benzyl, benzhydryl, trityl,9-phenylfluorenyl etc. A preferred nitrogen protecting group is Fmoc.

Sequences included to facilitate cloning or purification, etc., do notnecessarily contribute to the invention and may be omitted or removed.

It will be appreciated that sugar rings can exist in open and closedform and that, whilst closed forms are shown in structural formulaeherein, open forms are also encompassed by the invention.

MODES FOR CARRYING OUT THE INVENTION

ΔG287-953 hybrid protein

DNA encoding protein 287 from meningococcal serogroup B strain 394/98and protein 953 from meningococcal serogroup B strain 2996 were digestedand ligated, together with a short linker sequence, to give a plasmidencoding amino acid sequence SEQ ID 7. The plasmid was transfected intoE. coli and bacteria were grown to express the protein.

After adequate growth, bacteria were harvested and the protein waspurified. From culture, bacteria were centrifuged and the pellet washomogenized in the presence of 50 mM acetate buffer (pH 5) with apellet:buffer volume ratio of 1:8. Lysis was performed using a highpressure homogenizer (AVESTIN, 4 cycles at 14000 psi). After lysis, ureawas added at final concentration of 5M, followed by agitation for 1 hourat room temperature. The pH was reduced from 6 to 5 using 200 mM acetatebuffer (pH 4)+5 M urea. The mixture was centrifuged at 16800 g for 60minutes at 2-8° C. The supernatant was collected and filtered bySARTOBRAN P (0.45-0.22 μm SARTORIUS).

Protein in the filtered supernatant was stable for ≧30 days at −20° C.and for ≧15 days at 2-8° C.

Protein was further purified on a cationic exchange column (SPFF,Amersham Biosciences) with elution using 350 mM NaCl+50 mM acetate+5 Murea pH 5.00. The majority of impurities were present in the flow-thru.A pre-elution washing using a lower NaCl concentration (180 mM)advantageously eliminated two contaminating E. coli proteins.

The eluted material was adjusted to pH 8 (using 200 mM TRIS/HCl+5 M ureapH 9) and further purified on a Q Sepharose HP column (Amersham) withelution using 150 mM NaCl+20 mM TRIS/HCl pH 8.00 in 5 M urea. Again, apre-elution washing with reduced salt (90 mM) was useful for eliminatingimpurities.

The filtered eluted material from Q HP column was diluted 1:2 using PBSpH 7.00 (150 mM NaCl+10 mM potassium phosphate, pH 7.00) and thendiafiltered against 10 volumes of PBS pH 7.00 by tangentialultrafiltration. At the end of diafiltration the material wasconcentrated 1.6 times to about 1.2 mg/ml total proteins. Using a 30,000Da cut-off membrane (Regenerated Cellulose membrane 50 cm², MilliporePLCTK 30) it was possible to dialyze the material with a yield of about90%.

936-ΔG741 Hybrid Protein

DNA encoding protein 936 from meningococcal serogroup B strain 2996 andprotein 741 from meningococcal serogroup B strain MC58 were digested andligated, together with a short linker sequence, to give a plasmidencoding amino acid sequence SEQ ID 8. The plasmid was transfected intoE. coli and bacteria were grown to express the protein. The recombinantprotein was not secreted, but remained soluble within the bacteria.

After adequate growth, bacteria were centrifuged to give a humid pasteand treated as follows:

-   -   Homogenisation by high pressure system in presence of 20 mM        sodium phosphate pH 7.00.    -   Centrifugation and clarification by orthogonal filtration.    -   Cationic column chromatography (SP Sepharose Fast Flow), with        elution by 150 mM NaCl in 20 mM sodium phosphate pH 7.00.    -   Anionic column chromatography (Q Sepharose XL) with flow-through        harvesting.    -   Hydrophobic column chromatography (Phenyl Sepharose 6 Fast Flow        High Sub) with elution by 20 mM sodium phosphate, pH 7.00.    -   Diafiltration against PBS pH 7.4 with a 10 Kd cut-off.    -   Final sterile filtration and storing at −20° C.

Protein in the final material was stable for at least 3 months both at−20° C. and at 2-8° C.

NadA^((NL)(C)) Protein

DNA encoding NadA protein from meningococcal serogroup B strain 2996 wasdigested to remove the sequence encoding its C-terminus, to give aplasmid encoding amino acid sequence SEQ ID 1. The plasmid wastransfected into E. coli and bacteria were grown to express the protein.The recombinant protein was secreted into the culture medium, and theleader peptide was absent in the secreted protein (SEQ ID 2). Thesupernatant was treated as follows:

-   -   Concentration 7× and diafiltration against buffer 20 mM TRIS/HCl        pH7.6 by cross flow UF (Cut off 30 Kd).    -   Anionic column chromatography (Q Sepharose XL), with elution by        400 mM NaCl in 20 mM TRIS/HCl pH 7.6.    -   Hydrophobic column chromatography step (Phenyl Sepharose 6 Fast        Flow High Sub), with elution by 50 mM NaCl in TRIS/HCl pH 7.6.    -   Hydroxylapatite ceramic column chromatography (HA Macro. Prep)        with elution by 200 mM sodium phosphate pH 7.4.    -   Diafiltration (cut off 30 Kd) against PBS pH 7.4    -   Final sterile filtration and storing at −20° C.

Protein in the final material was stable for at least 6 months both at−20° C. and at 2-8° C.

NadA protein is susceptible to degradation, and truncated forms of NadAmay be detected by western blot or by mass spectrometry (e.g. byMALDI-TOF) indicating up to 10 kDa MW loss. Degradation products can beseparated from native NadA by gel filtration (e.g. using column TSK300SWXL, precolumn TSKSWXL, TOSOHAAS). Such filtration gives threepeaks: (i) a first peak with retention time 12.637 min and apparent MW885.036 Da; (ii) retention time 13.871 min and apparent MW 530.388 Da;(iii) retention time 13.871 min and apparent MW 530.388 Da. Lightscattering analysis of the three peaks reveals real MW values of (i)208500 Da, (ii) 98460 Da, (iii) 78760 Da. Thus the first peak containsNadA aggregates, and the third peak contains degradation products.

As the predicted molecular weight of NadA^((NL)(C)) is 34.113 Da, peak(ii) contains a trimeric protein, which is the desired antigen.

Antigenic Combinations

Mice were immunised with a composition comprising the three proteins andan aluminium hydroxide adjuvant. For comparison purposes, the threeproteins were also tested singly. Ten mice were used per group. Themixture was able to induce high bactericidal titres against variousstrains:

Meningococcal strain^((Serogroup)) 2996^((B)) MC58^((B)) NGH38394/98^((B)) H44/76^((B)) F6124^((A)) BZ133^((C)) C11^((C)) (1) 3200016000 130000 16000 32000 8000 16000 8000 (2) 256 131000 128 16000 320008000 16000 <4 (3) 32000 8000 — — — 8000 — 32000 Mix 32000 32000 6500016000 260000  65000 >65000 8000 ‘—’ indicates that this strain containsno NadA gene

Looking at individual mice, the triple mixture induced high andconsistent bactericidal titres against the three serogroup B strainsfrom which the individual antigens are derived:

# 1 2 3 4 5 6 7 8 9 10 2996 32768 16384 65536 32768 32768 65536 6553632768 65536 8192 MC58 65536 32768 65536 65536 65536 8192 65536 3276832768 65536 394/98 65536 4096 16384 4096 8192 4096 32768 16384 819216384Combination and Comparison with OMVs

In further experiments, the adjuvanted antigens (20 μg of each antigenper dose) were administered in combination with 10 μg OMVs preparedeither from strain H44/76 (Norway) or strain 394/98 (New Zealand).Positive controls were the anti-capsular SEAM-3 mAb for serogroup B orCRM197-conjugated capsular saccharides for other strains. Results(bactericidal titres) are shown in Table 1. The mixture almost alwaysgives better titres than simple OMVs and, furthermore, the addition ofthe mixture to OMVs almost always significantly enhances the efficacy ofthe OMVs. Moreover, in many cases the antigen mixture matches or exceedsthe response seen with the positive control.

Hypervirulent Lineage Tests

The following antigens were tested against a variety of serogroup Bstrains from a variety of hypervirulent lineages:

-   -   (a) NadA^((NL)(C))    -   (b) ΔG287-953    -   (c) 936-ΔG741    -   (d) a mixture of (a), (b) and (c)    -   (e) OMVs prepared from strain H44/76 (Norway)    -   (f) OMVs prepared from strain 394/98 (New Zealand)    -   (g) A mixture of ΔG287 and (e)    -   (h) A mixture of (d) and (e)    -   (i) A mixture of (d) and (f)

SEAM-3 was used as a positive control.

Results were as follows, expressed as the percentage of strains in theindicated hypervirulent lineage where the serum bactericidal titreexceeded 1024:

# strains (a) (b) (c) (d) (e) (f) (g) (h) (i) S-3 A4 4 50 50 0 100 25 2525 100 100 + ET-5 8 25 75 88 100 71 14 71 100 100 + Lineage 13 0 75 1593 8 85 8 92 93 + 3 ET-37 4 11 22 0 33 0 0 0 22 25 +

Against particular reference strains, bactericidal titres were asfollows:

Strain (a) (b) (c) (d) (e) (f) (g) (h) (i) S-3 A4 961-5945 128 2048 <82048 262144 8192 262144 262144 4096 8192 ET-5  44/76 <4 2048 32768131072 524288 8192 524288 524288 524288 16384 Lineage 3 394/98 <4 102432 4096 <4 16384 256 16384 16384 16384 ET-37 LPN17592 2048 1024 256 4096<8 <8 512 16384 65536 1024

Compositions (d), (h) and (i) therefore induce bactericidal antibodyresponses against a wide variety of strains of serogroup B.meningococcus from within hypervirulent lineages A4, ET-5 and lineage 3.Titres using compositions (h) and (i) were generally higher than with(d), but the coverage of strains within hypervirulent lineages A4, ET-5and lineage 3 were no better.

Coverage of untyped strains was also high with compositions (d), (h) and(i).

Analysis of NadA N-Terminus Domain

Purified N. meningitidis NadA protein is known to bind to humanepithelial cells [17] (e.g. Chang cells, HeLa cells, Hep-2 cells), andrecombinant E. coli which express NadA display an adherent phenotype[18]. These E. coli are also able to invade epithelial cells, andintracellular NadA^(+ve) E. coli can be detected in Chang cells byimmunofluorescence (after membrane permeabilisation) and by electronmicroscopy. NadA is thus believed function as an adhesin and an invasinfor epithelial cells.

On the basis of secondary structure analysis, mature NadA has beensubdivided into three putative domains: a N-terminal globular domain (aa24-87), an α-helix internal region (aa 88-350) with high coiled-coilpropensity, and a C-terminal membrane anchor (aa 351-405). The role ofthe N-terminal globular domain in host-cell interaction wasinvestigated.

A truncated nadA gene coding for a protein devoid of amino acids 30-87was cloned into pET-21 vector (pET-NadAA30-87) and expressed in E. coliBL21(DE3) strain. Amino acids 24-29 were retained to allow processing ofthe leader peptide and correct maturation of the protein. Western blotand FACS analysis confirmed that NadAA30-87 was expressed and formedoligomers on the E. coli cell surface i.e. deletion of the N-terminaldomain does not interfere with the expression, export and membranelocalization of NadA. However, the recombinant E. coli strain completelylost the capacity to adhere to Chang epithelial cells. The N-terminusdomain is thus implicated in adhesin activity.

To further investigate which part of the N-terminal domain is involvedin the interaction, the region was additionally divided into threeputative sub-domains: amino acids 24-42, containing a predicted α-helixregion with hydrophobic residues; amino acids 43-70, the internal partwithout a predicted defined secondary structure; and amino acids 71-87containing an other predicted α-helix structure. Three constructs, eachencoding a protein deleted of a single sub-domain, were generated andthen introduced into E. coli BL21(DE3), obtaining the following strains:BL21(DE3)/pET-NadAΔ24-42, BL21(DE3)/pET-NadAA43-70 andBL21(DE3)/pET-NadAΔ71-87. Surface localisation of the oligomers wasconfirmed by western blot and FACS analysis, but adhesion to Changepithelial cells was no better than the control BL21(DE3)/pET E. colistrain. These results, confirmed also using immunofluorescencemicroscopy analysis, indicate that the entire globular N-terminal domainof NadA is important in the interaction with human cells.

Combination with Meningococcal and/or Hib Conjugates

The triple MenB composition is combined with a mixture ofoligosaccharide conjugates for serogroups C, W135 and Y, to give avaccine containing the following antigens:

Component Quantity per 0.5 ml dose Serogroup C conjugate 10 μgsaccharide + 12.5-25 μg CRM₁₉₇ Serogroup W135 conjugate 10 μgsaccharide + 6.6-20 μg CRM₁₉₇ Serogroup Y conjugate 10 μg saccharide +6.6-20 μg CRM₁₉₇ ΔG287-953 20 μg polypeptide 936-ΔG741 20 μg polypeptideNadA 20 μg polypeptide

A similar vaccine is prepared, including MenA conjugate (10 μgsaccharide+12.5-33 μg CRM₁₉₇) and/or a HbOC Hib conjugate (10 μgsaccharide+2-5 μg CRM₁₉₇).

Use of Modified MenA Saccharide

Capsular polysaccharide was purified from MenA and was hydrolysed togive MenA oligosaccharide. The polysaccharide (2 g) was hydrolyzed at50° C. in 50 mM sodium acetate buffer, pH 4.75, at a Polysaccharideconcentration of 10 mg/mL for about 4 hours [135]. After hydrolysis, thesolution was dried by rotary evaporation.

The oligosaccharide was activated using the following reaction scheme:

The oligosaccharide was dissolved in DMSO to give a saccharideconcentration of 10 mg/mL. According to a molar ratio ofoligosaccharide:CDI being 1:20, 21.262 g of CDI was then added and thereaction mixture stirred for 16 hours at room temperature. The resultingMenA-CDI compound was purified by selective precipitation in a 80:20(v/v) acetone:DMSO mixture followed by centrifugation. The efficiency ofthe activation reaction was calculated to be about 67.9% by determiningthe ratio of free imidazole to bonded imidazole.

In the second reaction step, the MenA-CDI oligosaccharide wassolubilised in DMSO at a saccharide concentration of about 10 mg/mL.According to a molar ratio of MenA-CDI unit:DMA being 1:100, 36.288 g of99% dimethylamine hydrochloride (i.e. R¹ & R²=Me) was added and thereaction mixture stirred for 16 hours at room temperature. The reactionproduct was freeze-dried and re-solubilised in 10 mg/mL water solution.

To remove the low molecular weight reaction reagent (in particular thedimethylamine (DMA)) from the oligosaccharide preparation, a dialysisstep was performed through a 3.5 kDa MWCO membrane (Spectra/Por™). Fourdialysis steps were carried out: (i) 16 hours against 2 L of 1 M sodiumchloride (dialysis factor 1:20), (ii) 16 hours against 2 L of 0.5 Msodium chloride (dialysis factor 1:20), (iii) and (iv) 16 hours against2 L of WFI (dialysis factor 1:20). To improve the purification adiafiltration step was also performed through a 1 kDa MWCO membrane(Centricon™).

The purified MenA-CDI-DMA product was buffered at pH 6.5 in 25 mML-histidine (Fluka™).

For preparing conjugates of the modified MenA saccharide (MenA-CDI-DMA),the overall process was as follows:

-   -   hydrolysis of the polysaccharide to give oligosaccharide        fragments    -   sizing of the oligosaccharide fragments    -   reductive amination of terminal aldehyde groups on the sized        oligosaccharides    -   protection of terminal —NH₂ groups by Fmoc group before the CDI        reaction    -   intrinsic de-protection of —NH₂ groups during the DMA reaction    -   activation of terminal —NH₂ groups by SIDEA        (N-hydroxysuccinimide adipic acid)    -   covalent attachment to CRM₁₉₇ protein

The modified MenA oligosaccharide conjugate is much more resistant tohydrolysis than its natural counterpart at elevated temperatures. After28 days at 37° C., for instance, the percentage of released saccharideis 6.4% for the modified oligosaccharide vs. 23.5% for the naturalantigen. Moreover, the titres induced by the modified oligosaccharidesare not significantly lower than those obtained using the native sugarstructures.

The modified MenA conjugate is combined with MenC, MenW135 and MenYconjugates as a substitute for the conjugate of unmodifiedoligosaccharide. This tetravalent mixture is mixed with the three MenBpolypeptides to give a vaccine effective against serogroups A, B, C,W135 and Y of N. meningitidis in a single dose.

Pneumococcal Combinations

The three combined MenB proteins are mixed with pneumococcal saccharideconjugates to give a final concentration of 2 μg/dose of each of thepneumococcal serotypes (double for serotype 6B). The reconstitutedvaccine thus contains the following antigens:

Component Quantity per 0.5 ml dose Serogroup A conjugate 5 μgsaccharide + 6.25-16.5 μg CRM₁₉₇ Serogroup C conjugate 5 μg saccharide +6.25-12.5 μg CRM₁₉₇ Serogroup W135 conjugate 5 μg saccharide + 3.3-10 μgCRM₁₉₇ Serogroup Y conjugate 5 μg saccharide + 3.3-10 μg CRM₁₉₇Pneumococcus serotype 4 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 9V 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 14 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 18C 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 19F 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 23F 2 μg saccharide + 2.5 μg CRM₁₉₇ conjugatePneumococcus serotype 6B 4 μg saccharide + 5 μg CRM₁₉₇ conjugate

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

TABLE 1 2996 NGH38 M4215 MC58 44/76 CU385 N44/89 394/98 M01-240149Typing: B:2b:P1.5a,2a B:NT:P1.3 B:15:P1.7,16 B:15:P1.7,16b B:15:P1.7,16B:4:P1.15 B:4,7:P1.19,15 B:4:P1.4 B:4:P1.7,4 ET: other other n.d. ET5ET5 ET5 ET5 lin.3 lin.3 Positive 32768 32768 32768 16384 16384 >163848192 16384 8192 control Antigen 4096 4096 65536 32768 65536 >65536 >40968192 2048 mixture Antigens + H44/76 16384 8192 >65536 32768524288 >65536 >4096 16384 8192 OMVs Antigens + 394/98 8192 8192 >6553632768 >65536 >65536 >4096 65536 >8192 OMV OMVs (Norway) <4 1024 81922048 262144 256 <8 4096 <4 OMVs (NZ) 512 <4 128 2048 <4 <8 <832768 >8192 NM092 NM008 BZ198 961-5945 G2136 5/99 F6124 BZ133 LPN17592240539 Typing: B:4:P1.4 B:4:P1.4 B:NT B:2b:P1.21,16 B: B:2b:P1.5,2 AC:NT: W135 P1.5 ET: lin 3 lin 3 lin 3 A4 A4 A4 slll sl Positive 327688192 16384 8192 32768 8192 1024 1024 4096 control Antigen >4096 40964096 2048 2048 >4096 8192 16384 4096 >8192 mixture Antigens +H44/76 >4096 >4096 >4096 >8192 2048 >4096 32768 32768 16384 >8192 OMVsAntigens + 394/98 >4096 >4096 >4096 2048 8192 >4096 65536 6553665536 >8192 OMV OMVs (Norway) <8 <8 <4 >8192 <8 <8 1024 <4 <8 >4096 OMVs(NZ) 4096 1024 4096 <16 n.d. <8 4096 1024 <8 >4096

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1. A composition which, after administration to a subject, is able toinduce an antibody response in that subject, wherein the antibodyresponse is bactericidal against two or more of hypervirulent lineagesA4, ET-5 and lineage 3 of N. meningitidis serogroup B.
 2. Thecomposition of claim 1, comprising from 2 to 10 polypeptides, eachhaving a different amino acid sequence.
 3. The composition of claim 1,wherein the components which give rise to the bactericidal antibodyresponse are obtained by recombinant expression.
 4. The composition ofclaim 1 comprising five meningococcal antigens: (1) a ‘NadA’ protein;(2) a ‘741’ protein; (3) a ‘936’ protein; (4) a ‘953’ protein; and (5) a‘287’ protein.
 5. The composition of claim 4, wherein the NadA proteinhas 85% or more identity to SEQ ID NO:
 2. 6. (canceled)
 7. Thecomposition of claim 4, wherein the 741 protein has 85% or more identityto SEQ ID NO:
 3. 8. (canceled)
 9. The composition of claim 4, whereinthe 936 protein has 85% or more identity to SEQ ID NO:
 4. 10. (canceled)11. The composition of claim 4, wherein the 953 protein has 85% or moreidentity to SEQ ID NO:
 5. 12. (canceled)
 13. The composition of claim 4,wherein the 287 protein has 85% or more identity to SEQ ID NO:
 6. 14.(canceled)
 15. The composition of claim 4, wherein at least two of theantigens (1) to (5) are expressed as a single polypeptide chain.
 16. Thecomposition of claim 1, wherein the composition comprises a polypeptidewhich comprises a pair of antigens within a single polypeptide chainselected from the group consisting of: NadA & 741; NadA & 936; NadA &953; NadA & 287; 741 & 936; 741 & 953; 741 & 287; 936 & 953; 936 & 287;953 &
 287. 17. The composition of a claim 1, wherein the compositioncomprises a polypeptide of formula NH₂-A-[-X-L-]_(n)-B—COOH, wherein: Xis an amino acid sequence of one of the five antigens (1) to (5); L isan optional linker amino acid sequence; A is an optional N-terminalamino acid sequence; B is an optional C-terminal amino acid sequence;and n is 2, 3, 4, or
 5. 18. The composition of claim 17, wherein n is 2,X₁ is a 936 protein and X₂ is a 741 protein.
 19. The composition ofclaim 17, wherein n is 2, X₁ is a 287 protein and X₂ is a 953 protein.20. The composition of claim 1, comprising a protein comprising SEQ IDNO:
 7. 21. The composition of claim 1, comprising a protein comprisingSEQ ID NO:
 8. 22. The composition of claim 1, further comprisingsaccharide antigens from meningococcus serogroups Y, W135, C and(optionally) A.
 23. The composition of claim 1, further comprising asaccharide antigen from Haemophilus influenzae type B.
 24. Thecomposition of claim 22, wherein the saccharide antigen is conjugated toa carrier selected from: diphtheria toxoid, tetanus toxoid, CRM₁₉₇ or H.influenzae protein D.
 25. The composition of claim 1, further comprisingan antigen from Streptococcus pneumoniae. 26-27. (canceled)
 28. A methodfor raising an antibody response in a mammal, comprising the step ofadministering an effective amount of a composition of claim
 1. 29. Apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1 to
 8. 30. A process for purifying solubleNadA from a culture medium, comprising the steps of: concentration anddiafiltration against a buffer by ultrafiltration; anionic columnchromatography; hydrophobic column chromatography; hydroxylapatiteceramic column chromatography; diafiltration against a buffer; andfilter sterilisation.
 31. A process for purifying a 936-ΔG741 hybridprotein from a bacterium, comprising the steps of: homogenisation;centrifugation; cationic column chromatography; anionic columnchromatography; hydrophobic column chromatography; diafiltration againsta buffer; and filter sterilisation.